Molecular biology and pharmaceutical drug development now make intensive use of nucleic acid analysis. The most challenging areas are whole genome sequencing, single nucleotide polymorphism detection, screening and gene expression monitoring, which typically require analysis of large amounts of nucleic acid.
One area of technology which revolutionised the study of nucleic acids was the development of nucleic acid amplification techniques, such as the polymerase chain reaction (PCR). Amplification reactions, such as PCR, can enable the user to specifically and selectively amplify a particular target nucleic acid of interest from a complex mixture of nucleic acids. However, there is also an ongoing need for nucleic acid amplification techniques which enable simultaneous amplification of complex mixtures of templates of diverse sequence, such as genomic DNA fragments (e.g. “whole genome” amplification) or cDNA libraries, in a single amplification reaction.
PCR amplification cannot occur in the absence of annealing of forward and reverse amplification primers to primer binding sequences in the template to be amplified under the conditions of the annealing steps of the PCR reaction, i.e. if there is insufficient complementarity between primers and template. Some prior knowledge of the sequence of the template is therefore required before one can carry out a PCR reaction to amplify a specific template, unless random primers are used with a consequential loss of specificity. The user must usually know the sequence of at least the primer-binding sites in the template in advance so that appropriate primers can be designed, although the remaining sequence of the template may be unknown. The need for prior knowledge of the sequence of the template increases the complexity and cost of PCR amplification of complex mixtures of templates, such as genomic DNA fragments.
WO 98/44151 and WO 00/18957 both describe methods of forming polynucleotide arrays based on “solid-phase” nucleic acid amplification wherein the amplification products are immobilised on a solid support in order to form arrays comprised of nucleic acid clusters or “colonies”. Each cluster or colony on such an array is formed from a plurality of identical immobilised polynucleotide strands and a plurality of identical immobilised complementary polynucleotide strands. The arrays so-formed are generally referred to herein as “clustered arrays” and their general features will be further understood by reference to WO 98/44151 or WO 00/18957, the contents of both documents being incorporated herein in their entirety by reference.
As aforesaid, the solid-phase amplification methods of WO 98/44151 and WO 00/18957 are essentially carried out on a solid support. Like any amplification reaction these methods require the use of forward and reverse amplification primers (which may be identical or different) capable of annealing to a template to be amplified. In the methods of WO 98/44151 and WO 00/18957 both primers are immobilised on the solid support at the 5′ end. Other forms of solid-phase amplification are known in which only one primer is immobilised and the other is present in free solution (Mitra, R. D and Church, G. M., Nucleic Acids Research, 1999, Vol. 27, No. 24).
In common with all amplification techniques, solid-phase PCR amplification requires the use of forward and reverse amplification primers which include “template-specific” nucleotide sequences which are capable of annealing to sequences in the template to be amplified, or the complement thereof, under the conditions of the annealing steps of the amplification reaction. The sequences in the template to which the primers anneal under conditions of the amplification reaction may be referred to herein as “primer-binding” sequences.
Certain embodiments of the methods described in WO 98/44151 and WO 00/18957 make use of “universal” primers to amplify templates comprising a variable target portion that it is desired to amplify flanked 5′ and 3′ by common or “universal” primer binding sequences. The “universal” forward and reverse primers include sequences capable of annealing to the “universal” primer binding sequences in the template construct. The variable target portion may itself be of known, unknown or partially known sequence. This approach has the advantage that it is not necessary to design a specific pair of primers for each target to be amplified; the same primers can be used for amplification of different targets provided that each target is modified by addition of the same universal primer-binding sequences to its 5′ and 3′ ends. The variable target sequence can therefore be any DNA fragment of interest. An analogous approach can be used to amplify a mixture of targets, such as a plurality or collection of target nucleic acid molecules (e.g. genomic DNA fragments), using a single pair of universal forward and reverse primers, provided that each target molecule in the collection is modified by the addition of the same universal primer-binding sequences.
Such “universal primer” approaches to PCR amplification, and in particular solid-phase PCR amplification, are advantageous since they enable multiple template molecules of the same or different, known or unknown sequence to be amplified in a single amplification reaction, which may be carried out on a solid support bearing a single pair of “universal” primers. Simultaneous amplification of a mixture of templates of different sequences by PCR would otherwise require a plurality of primer pairs, each pair being complementary to each unique template in the mixture. The generation of a plurality of primer pairs for each individual targets is not a viable option for complex mixtures of targets.
The addition of universal priming sequences onto the ends of targets to be amplified can be achieved by a variety of methods known to those skilled in the art. For example, a universal primer consisting of a universal sequence at its 5′ end and a degenerate sequence at its 3′ end can be used in a PCR (DOP-PCR, eg PNAS 1996 vol 93 pg 14676-14679) to amplify fragments randomly from a complex target or a complex mixture of targets. The degenerate 3′ portion of the primer anneals at random positions on DNA and can be extended to generate a copy of the template that has the universal sequence at its 5′ end.
Alternatively, adapters that contain universal priming sequences can be ligated onto the ends of targets. The adapters may be single-stranded or double-stranded. If double-stranded, they may have overhanging ends that are complementary to overhanging ends on the target molecules that have been generated with a restriction endonuclease. Alternatively, the double-stranded adapters may be blunt, in which case the targets are also blunt ended. The blunt ends of the targets may have been formed during a process to shear the DNA into fragments, or they may have been formed by an end repair reaction, as would be well known to those skilled in the art. The ends of the targets may be treated to obtain a single 3′-overhang.
A single adapter or two different adapters may be used in a ligation reaction with targets. If a target has been manipulated such that its ends are the same, i.e. both are blunt or both have the same overhang, then ligation of a single compatible adapter will generate a target sequence with that adapter on both ends. However, if two compatible adapters, adapter A and adapter B, are used, then three permutations of ligated products are formed: target with adapter A on both ends, target with adapter B on both ends, and target with adapter A on one end and adapter B on the other end. This last product is, under some circumstances, the only desired product from the ligation reaction and consequently additional purification steps are necessary following the ligation reaction to purify it from the ligation products that have the same adapter at both ends.